Recent estimates indicate that about 85% of mutations occurring in the human genome are point mutations, that is they involve a very short sequence of nucleotides. Such mutations typically comprise substitutions of at least one nucleotide for another. This is in contrast to grosser changes such as deletions, insertions, inversions or duplications of longer DNA sequences. Current detection procedures capable of detecting single base substitutions include procedures based on differential denaturation of mismatched probes, as in allele specific oligonucleotide hybridization (Wallace, R. B., et al. (1979), Nucl. Acids Res., 6, 3553) or denaturing gradient gel electrophoresis (Myers, R. M., et al. (1985), Nature. 313, 495). Alternately, the sequence of interest can be investigated for abberations using restriction enzymes as in RFLP analysis (Geever, R. F. (1987), Proc. Natl. Acad. Sci. USA. 78, 508) or using RNAse A to cleave a mismatched nucleotide of an RNA probe hybridized to a target molecule (Myers, R. M., et al. (1985), Science, 230, 1242; Winter, E., et al. (1985), Proc. Natl. Acad. Sci. USA, 82, 7575). While the two techniques using denaturing gradient gels or RNAse A have the advantage of surveying long stretches of DNA for mismatched nucleotides, they are estimated to detect only about half of all mutations involving single nucleotides. Similarly, it is suggested that only approximately one third of the nucleotides in the human genome can be analyzed as part of restriction enzyme recognition sequences. The only existing technique capable of identifying any single nucleotide differences, short of sequencing, is allele specific oligonucleotide hybridization. This technique involves immobilizing separated (Wallace, supra) or enzymatically amplified fragments of test DNA (Saiki, R. K., et al. (1986), Nature, 329, 166), hybridizing with oligonucleotide probes, and washing under carefully controlled conditions to discriminate single nucleotide mismatches.
Whiteley, N. M. et al., EPO Publication No. 0 185 494, discloses an assay in which target sequences in nucleic acids are identified using two oligonucleotide probes of different length (e.g. a 15-mer and a 65-mer), selected to hybridize to contiguous regions of a target nucleic acid which has been previously immobilized. One of the probes is labeled. The short probe includes a potential mutation. Hybridization and/or washing is stated to be performed under conditions of stringency so specific that one probe complementary to a particular sequence will not hybridize (or remain hybridized during high stringency washes) if it is mismatched in one position. If hybridization of both probes occurs under high stringency conditions, the two probes are joined by ligation. Hybridization and ligation is determined by detecting whether label is incorporated in a ligated product. The presence of the label is stated to be an indication of the matching, at the selected stringency, of the short probe with the target nucleic acid sequence. However, this technique is subject to imprecision in that the stringency conditions of hybridizing are difficult to adjust so accurately that they can distinguish hybridization differences based upon a single base pair mismatch.
The references discussed above are provided solely for their disclosure prior to the filing of the instant case, and nothing herein is to be construed as an admission that such references are prior art or that the inventors are not entitled to antedate such disclosure by virtue of prior invention or priority based on earlier filed applications.